Recurrent emergence of Klebsiella pneumoniae carbapenem resistance mediated by an inhibitory ompK36 mRNA secondary structure

Significance Carbapenem-resistant Klebsiella pneumoniae represents an urgent threat to human health. Together with carbapenemase-mediated hydrolysis, mutations in the outer membrane porin OmpK36 have evolved to limit carbapenem influx. Analysis of the ompK36 gene from high-risk K. pneumoniae sequence types revealed the repeated emergence of an identical 5′ synonymous mutation. Whilst synonymous mutations are usually considered silent, we show that it reduces OmpK36 translation by inducing the formation of a messenger RNA secondary structure that obstructs the ribosomal binding site. While OmpK36 depletion attenuates virulence in a mouse lung infection model, it tips the balance towards antibiotic therapy failure. These results show mechanistically how the de novo emergence of a synonymous mutation contributes to last line antimicrobial resistance.


Materials and Methods
General cloning, molecular biology and strain generation All in vitro assays and animal infections with KP were carried out using ICC8001, a strain derived by animal passage from ATCC43816(1).
A homologous recombination technique resulting in scarless markerless mutants was used to generate all strains based on the Standard European Vector Architecture platform pSEVA612S (JX560380.2) mutagenesis plasmid. Briefly, mutants were generated in two sequential recombination steps. The first step integrates the mutagenesis plasmid into the genome generating a merodiploid. The mutagenic plasmid was introduced by three part  wild-type ORF (+500bp flanking regions) was cloned from ICC8001 genomic DNA with primers P7/P8 and ligated by Gibson assembly into pSEVA612S, linearised with P3/P4. In screening colonies by Sanger Sequencing we found the 25c>t mutation and this was not generated by site directed mutagenesis.

sfGFP vectors and -14a>t vectors
Leucine 9 CTG-sfGFP (wild-type) and Leucine 9 TTG-sfGFP (25c>t transition). The wild-type and 25c>t Sec signal sequences were cloned from the ompK36 vectors generated above with P9/P10. sfGFP was amplified from our previously published glmS site insertion vector (Vector 13 in Wong et al., 2019) with P11/P12 and chimeric fusion generated in the mutagenesis vector ligated by Gibson Assembly.
The 24c>t mutation was introduced by SDM using primers P31 and P32.

Introduction of pKpQIL KPC-2 plasmid
We introduced pKpQIL KPC-2 by conjugation using an E. coli donor following our previously published protocol(1).

Outer membrane preparations and gel electrophoresis
Outer membrane proteins were purified according to a previously described protocol with several modifications (3). Saturated overnight cultures of bacteria were grown in LB (10g/L NaCl) and sonification was performed at 25% amplitude for 10 bursts of 15 seconds each. 10 μg of protein was separated by SDS-PAGE using 12% acrylamide Mini-protean TGX precast gels (Bio-Rad, USA). Gels were stained with Coomassie (Sigma-Aldrich) and imaged on a ChemiDoc XRS+ (Biorad, USA).
RNA isolation and reverse transcription quantitative PCR (RT-qPCR) 300 µl of an LB overnight culture of KP were treated with RNAprotect Bacteria reagent (Qiagen) and centrifuged at 5000 xg for 10 min. The bacterial pellet was digested in 100 µl TE buffer with 15 mg/ml Lysozyme (Sigma-Aldrich) and 20 µl Proteinase K (Qiagen) for 10 min according to manufacturer's guidelines. RNA was isolated using the RNeasy minikit (Qiagen) following the manufacturer's instructions, and RNA concentration was determined using a Nanodrop 2000 spectrophotometer (ThermoFisher Scientific). 1 µg of RNA was treated with RNase-free DNase (Sigma-Aldrich) for 1 h at 37 °C and cDNA was then synthesized using a Moloney murine leukemia virus (MMLV) reverse transcription kit with random primers following the manufacturer´s protocol (Promega). To check for the presence of remaining DNA, a reaction mixture without the MMLV reverse transcriptase was also included (NRT). qPCR was performed using the Power Up SYBR Green master mix (ThermoFisher Scientific) and the following primers 33/34 (ompK36) and 35/36 (rpoB). The assay was run on a StepOnePlus System (Applied Biosystems) and results were analysed using the StepOne software (Applied Biosystems). Relative gene expression levels were analyzed by using the 2 −ΔCT (where CT is threshold cycle) method.

Housing and Randomisation
Upon arrival animals were independently randomized into cages of 5 animals and housed for an acclimatisation period of 1 week. Mice received food and water ad libitum and were housed in a 12hour/12hour light dark cycle. Identification of animals within groups was achieved by ear notching without anaesthesia at least 24 hours in advance of procedures.

Intratracheal administration of inoculum and infection
Anaesthesia was induced by intraperitoneal injection (BD Microlance 27G 13mm needle) of 80mg/kg ketamine and 0.8mg/kg medetomidine. Pre-intubation body temperature was maintained by contact with a heat mat (Harvard Apparatus, U.K) and eye lubricant (2.0mg/g carbomer, Alcon UK) was applied.
Intubation was achieved as previously described (1) At the experimental end-point animals were anesthetized with ketamine 100mg/kg and 1mg/kg medetomidine by intraperitoneal injection (BD Microlance 27G 13mm needles). Following the induction of anaesthesia, blood was collected by a transdiaphragmatic inferior approach cardiac puncture (BD Microlance 25G 25mm needles) and animals were then humanely killed, under anaesthesia, by cervical dislocation.
An aliquot of blood (20ul) was taken and diluted into 180ul of hypotonic lysis solution (1mM EDTA in water) for enumeration of bacterial counts. The rest was placed into a microtainer (BD Microtainer for serum collection, BD Medical) and allowed to clot for 1 hour. This was then spun according to the manufacturer's guidance and aliquoted into cryobanking tubes (Greiner Bio-One) and frozen at -80 °C until analysis.
Bacteria were enumerated from the lung homogenate and blood by dilution in PBS and plating onto solidified LB agar containing 50ug/ml rifampicin (Merck, UK).

Antibiotic delivery
We combined the meropenem (100mg/kg) with cilastatin (100mg/kg) (Merck, UK), a renal dihydropeptidase inhibitor, to reduce the in vivo metabolism of meropenem as the murine enzyme isoform has increased activity against this carbapenem compared to human enzyme.
Drugs were diluted in dH2O. dH2O alone was administered to vehicle control animals. Doses were delivered at 6 hourly intervals as previously employed in a murine carbapenemase producing KP model(4). The drug mixture was delivered by intraperitoneal infection (BD Microlance 27G 13mm needles) to animals at 6 hourly intervals and the iliac fossa used was alternated between doses.

Cytokine bead assay
Serum IFN-γ was assessed using a custom-made mouse panel 13-plex kit (LEGENDplex, BioLegend) following the manufacturer's instructions. Cytokine levels were acquired using a FACSCalibur flow cytometer (BD Biosciences), and analyses were performed using LEGENDplex data analysis software (BioLegend). A biological repeat was considered valid only if at least 80% of all the samples run were above the limit of detection; any values below the detection limit within a repeat such as this were assumed to be the lowest value detectable by the assay for statistical analysis. Values below the detection limit in graphs were displayed as 1 for visualisation.

Determination of RNA structures using DMS-MaPseq
In vitro DMS modification Plasmids encoding the wildtype and mutant ompk36 were isolated using miniprep (Zymo) and amplified by PCR using a forward primer containing the T7 promoter sequence (P37) and a reverse primer (P38). The PCR product was used for T7 Megascript in vitro transcription (ThermoFisher Scientific) according to manufacturer's instructions. Next, the RNA was purified using RNA Clean and Concentrator TM -5 (Zymo). 10µg RNA was denatured at 95°C for 1 min. Briefly, RNA was fragmented using manufacturer's instructions for 2 minutes without adding reagent F2 (dNTPs). After 2 minutes, fragmentation mix was placed on ice immediately. The mixture was then added with the TGIRT reverse transcriptase mix, which consists of 1μl TGIRT, 1μl water, 1μl enzyme R1 (RNAse inhibitor) and 1μl DTT, and incubated at room temperature for 30 minutes. Then, F2 (dNTPs) was added and the fragmented RNA mixture was reverse transcribed under the conditions: 20°C for 10 mins, 42°C for 10 mins, 55°C for 60 mins, denaturation by adding 1μl 4M NaOH at 95°C for 3 mins. The mixture was neutralized by adding 2μl of 4M HCl and the volume of neutralized mixture brought up to 50μl. Then, the cDNA was cleaned up using 1x volume ratio of SPRI beads (Beckman Coulter) and eluted in 10μl EDTA TE. Samples were then adapted, extended, ligated and amplified for 10 cycles following IDT's instructions. The libraries (~300-400 bp) were gel-purified on an 8% TBE polyacrylamide gel (ThermoFisher Scientific) and precipitated using isopropanol. The libraries were then loaded on iSeq-100 sequencing flow cell with iSeq-100 High-throughput sequencing kit and library was run on iSeq-100 (paired-end run, 151 x 151 cycles).
In vivo DMS modification, total RNA extraction and rRNA subtraction For in vivo DMS modification, 500µl of exponentially growing E. coli were incubated with 10µl DMS for 3 mins at 37°C while shaking at 800 r.p.m. on a thermomixer. DMS was quenched by adding 500µl 30% BME, followed by 3 min 30% BME wash and 1x PBS wash. Then, bacterial pellets were resuspended and incubated at room temperature for 5 mins in 500µl RNAprotect® bacteria reagent (QIAGEN). Samples were centrifuged at 16000g for 5 mins and supernatant removed. Pellets were resuspended in 100µl of 15mg/ml lysozyme solution plus 20µl proteinase K and mixed by vortex for 10 seconds. Samples were incubated at room temperature for 10 mins, vortexing for 10 seconds every 2 mins. Next, samples were added with 350µl of buffer RLT plus BME (10µl BME for every 1ml of RLT), vortex to mix. Then, 250µl of 96-100% ethanol was added to samples. RNA was extracted using the RNeasy® Mini kit (QIAGEN) following manufacturer's instructions. DNA was digested from 5-10µg of total RNA per sample using the TURBO DNA-free kit (ThermoFisher Scientific) and purified using RNA Clean and Concentrator TM -5 kit (Zymo). Following that, ribosomal RNAs were depleted using the Ribominus TM Transcriptome Isolation kit for bacteria (ThermoFisher Scientific) following manufacturer's instructions. RNA was purified using RNA Clean and Concentrator  and eluted in 10µl water.

DMS-MaPseq library generation of target sequence-specific in vivo DMS-modified RNA
To reverse transcribe, rRNA-depleted total RNA purified from the previous steps was added to 4µl 5x FS buffer, 1µl dNTP, 1µl 0.1M DTT, 1µl RNase Out, 1µl 10uM reverse primer (P39) and 1µl TGIRT-III (Ingex). The reaction was incubated for 1.5h at 60°C. Then, to degrade the RNA, 1ul 4M NaOH was added and incubated for 3min at 95°C. The cDNA was purified in 10µl water using the Oligo Clean and Concentrator TM kit (Zymo). Next, 1µl of cDNA was amplified using Advantage HF 2 DNA polymerase (Takara) for 30 cycles according to the manufacturer's instructions (P40/P41). The PCR product was purified using E-Gel TM SizeSelect TM II 2% agarose gel (Invitrogen). RNA-seq library for 300bp insert size was constructed following the manufacturer's instructions (NEBNext Ultra TM II DNA Library Prep Kit). The library was loaded on iSeq-100 sequencing flow cell with iSeq-100 High-throughput sequencing kit and library was run on iSeq-100 (paired-end run, 151 x 151 cycles).

DMS-MaPseq analysis
To determine the DMS signal, FASTQ files were processed and analyzed using the DREEM (Detection of RNA folding Ensembles using Expectation-Maximization clustering) pipeline (5).
Briefly, reads were trimmed using TrimGalore (github.com/FelixKrueger/TrimGalore) to remove Illumina adapters. Trimmed paired reads were then mapped to ompk36WT (accession number CP009208.1, annotated as ompC4), 25c>t and double mutant using Bowtie2 with the parameters: --local --no-unal --no-discordant --no-mixed -L 12 -X 1000. A bit vector was generated for each pair of aligned reads and their mutational signatures were analyzed using the DREEM algorithm (5). The ratio of mismatches and deletions to total coverage at each nucleotide position was calculated to quantify the population average DMS reactivity at each position. Then, DMS reactivities were normalized to the median of the top 5% of DMS reactivities to a scale of 0 to 1. The normalized DMS signals were used as folding constraints for predicting RNA secondary structures with the program RNAstructure v. 6